The purification of biopharmaceutical molecules involves using chromatographic separation methods as one of wide established technologies. The usual application involves capture and polishing of the target molecule. Various chromatographic methods and resins can be applied to fulfill these tasks (e.g. normal phase, reversed phase, size exclusion and ion exchange chromatography modes). In special areas this has developed to more specific and more efficient separation methods such as affinity chromatography. The current state of art of the antibody purification is the use of ProtA modified affinity chromatography support materials to bind and separate the target molecule from the greater part of most abundant impurities (host cell proteins, host cell DNA, sugars, amino acids, growth factors and etc.). More than 30 chromatographic resins with various properties are available today in the market, providing an opportunity for the customer to find the one best suited to his application. The main differences in those products are physical (support material, particle size, particle shape and pore size) as well as chemical properties (ProtA ligand, ligand density, support matrix type) which influence the binding capacity, mass transfer properties, compressibility and robustness.
The binding capacity strongly depends on the affinity ligand, its density on the surface and its accessibility. Due to the improvements in the modification technologies, high binding capacities >30 g/L can be reached at residence times of >3 min independently from the support matrix chemistry.
On the other hand, mass transfer properties are quite variable, causing radical differences in the application. Film mass transfer and pore diffusion are two main parameters, which influence the target molecule diffusivity towards the adsorption sites. Film mass transfer is mainly influenced by the particle size and shape. The smaller the particle and the interparticle volume the greater the mass transfer and the flow resistance. Therefore, for the analytical scale applications smaller particle size is used to assure a fast mass transfer and high efficiency. But due to the pressure resistance issues, the industrial scale operations are performed at <3 bar operation pressures. Bigger particles (60 μm-120 μm) are usually chosen.
Due to the above mentioned issues, the pore diffusion is one of the most influential properties defining the mass transfer. It was shown that some materials exhibit wide pores (50-500 nm) to enable a fast mass transfer. But these materials show low binding capacities (˜20 g/L), due to the low surface area. To enhance binding capacity, higher surface area is required, usually achieved through smaller pore size. Materials exhibiting ˜100 nm pore sizes have shown 40 g/L (PROSEP®-vA High Capacity (Millipore Bioprocessing, Consett, UK) binding capacities, even more ˜70 nm pore size exhibiting materials have shown 56 g/L (PROSEP®-vA Ultra (Millipore Bioprocessing, Consett, UK) binding capacities. Support materials with wide pore size distribution in the comparable range have shown also similar binding capacities (MABSELECT SURE® (GE Healthcare, Uppsala, Sweden). Due to the smaller pore size, the diffusion of the target molecule is slower, requiring certain target molecule residence time to achieve binding capacities above 40 g/l. The usual range of used residence times is in the range 3-6 minutes to assure that the target molecule diffuses towards the binding sites. This is important in the chromatographic process loading phase, where the support material is treated with a solution containing target molecule. The rest of the necessary affinity chromatography method steps (such as wash, elution, Strip, cleaning in place, reequilibration, etc.) can be performed faster to assure a shorter processing time. The operational velocity in this case is influenced by the support material rigidity.
Nevertheless, after the application of this specific, very selective method for about 20 years, there is an increasing need for further method optimization in pursuit of higher economic efficiency and productivity (g (target molecule)/ml (stationary phase)/h (operation time). The current status is to use big columns for higher throughput in the expense of higher investment for the higher resin amounts. Sometimes columns of 1.2 meter diameter and 10-20 cm bed height are used for mAb purification. It was recognized that there is an alternative technology that might enable economic processing, namely continuous chromatography. The basics of this method is higher resin utilization while loading to 80% of resin binding capacity instead of 60% as in standard batch without product loss, shorter operation times through splitting the sequential batch process into the parallel operation on numerous columns and therefore enabling the usage of smaller columns.
In continuous chromatography, several identical columns are connected in an arrangement that allows columns to be operated in series and/or in parallel, depending on the method requirements. Thus, all columns can be run in principle simultaneously, but slightly shifted in method steps. The procedure can be repeated, so that each column is loaded, eluted, and regenerated several times in the process. Compared to “conventional” chromatography, wherein a single chromatography cycle is based on several consecutive steps, such as loading, wash, elution and regeneration, in continuous chromatography based on multiple identical columns all these steps occur simultaneously but on different columns each. Continuous chromatography operation results in a better utilization of chromatography resin, reduced processing time and reduced buffer requirements, all of which benefits process economy. One example of continuous chromatography is simulated moving bed (SMB) chromatography. In the current years this simulated moving bed (SMB) technology was introduced to the biopharmaceutical market as well, especially to the purification of antibodies. If previously in normal batch chromatography the loading of the chromatographic column was stopped at a specific percentage of target molecule breakthrough, so now the loading can be continued in order to increase the binding capacity without target molecule losses, since a brake through fraction is loaded on the column that is following the first one.
Nevertheless, for a better economic efficiency the processing time is of the major importance since it directly influences productivity. The increase in the binding capacity is already increasing the productivity by the level on how much more material can be bound to the surface (˜20-40%), but decreasing the residence time required for the target molecule to be bound on the modified surface would enhance the economic efficiency even more. Presumably, decreasing the residence time to 0.3 min will lead to 10 times increased productivity if compared to 3 min residence time. Unfortunately, decreasing the residence time during the target molecule adsorption phase leads to the decrease in the dynamic binding capacity since the rate of the mass transfer is decreased. Consequently, column loading is typically performed with velocities going up to about 600 cm/h as disclosed in US 20090209736. But as it is also actually stated in E. Mahajan et el., Journal of Chromatography A, 1227 (2012) 154-162, at higher flow rates the column binding capacity is reduced so that high flow rates are not feasible for column loading.
Surprisingly we found that an appropriate support material physical property (particle size, pore size) selection coupled with a certain continuous chromatography method enables loading velocities above 800 cm/h and more.